{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,22]],"date-time":"2026-10-22T16:41:07Z","timestamp":1792687267169,"version":"4.5.0"},"reference-count":47,"publisher":"Wiley","issue":"3","license":[{"start":{"date-parts":[[2025,11,4]],"date-time":"2025-11-04T00:00:00Z","timestamp":1762214400000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/onlinelibrary.wiley.com\/termsAndConditions#vor"},{"start":{"date-parts":[[2025,11,4]],"date-time":"2025-11-04T00:00:00Z","timestamp":1762214400000},"content-version":"tdm","delay-in-days":0,"URL":"http:\/\/doi.wiley.com\/10.1002\/tdm_license_1.1"}],"content-domain":{"domain":["analyticalsciencejournals.onlinelibrary.wiley.com"],"crossmark-restriction":true},"short-container-title":["Biotech &amp; Bioengineering"],"published-print":{"date-parts":[[2026,3]]},"abstract":"<jats:title>ABSTRACT<\/jats:title>\n                  <jats:p>\n                    Nicotinamide adenine dinucleotide (NAD\u207a) is an essential redox cofactor widely used in industrial biocatalysis and therapeutic applications. Conventional chemical synthesis of NAD\u207a is hazardous and inefficient, while enzymatic approaches, particularly dual\u2010enzyme cascades involving nicotinamide riboside kinase (NRK) and nicotinamide mononucleotide adenylyltransferase (NMNAT), suffer from low catalytic efficiency due to spatially separated active sites and poor intermediate channeling. Here, we report a direct, one\u2010step NAD\u207a biosynthetic route using\n                    <jats:italic>Haemophilus influenzae<\/jats:italic>\n                    NadR, a naturally bifunctional enzyme that naturally integrates NRK and NMNAT activities into a single polypeptide chain. To address the rate\u2010limiting NR phosphorylation step, we developed a loop engineering\u2013docking combinatorial simulation (LEDCS) strategy to rationally redesign the NRK domain. The resulting V241S\/L387Q mutant displayed a fourfold increase in enzymatic activity and a 2.4\u2010fold improvement in catalytic efficiency relative to the wild type. Structural and interaction analyses revealed that the P\u2010loop plays a critical role in coordinating NR and ATP binding, while enhanced hydrophilicity in the substrate\u2010binding pocket improved substrate affinity. This study highlights the catalytic advantage of intramolecular substrate channeling and presents a generalizable strategy for enhancing the performance of multifunctional biocatalysts.\n                  <\/jats:p>","DOI":"10.1002\/bit.70097","type":"journal-article","created":{"date-parts":[[2025,11,5]],"date-time":"2025-11-05T07:07:22Z","timestamp":1762326442000},"page":"627-639","update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":1,"title":["Structure Guided Engineering of Bifunction Enzyme NadR for Enhanced Nicotinamide Adenine Dinucleotide Production"],"prefix":"10.1002","volume":"123","author":[{"given":"Dianju","family":"Wei","sequence":"first","affiliation":[{"name":"Key Laboratory of Bioorganic Synthesis of Zhejiang Province, The National and Local Joint Engineering Research Center for Biomanufacturing of Chiral Chemicals, College of Biotechnology and Bioengineering Zhejiang University of Technology Hangzhou China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yinfeng","family":"Huang","sequence":"additional","affiliation":[{"name":"Key Laboratory of Bioorganic Synthesis of Zhejiang Province, The National and Local Joint Engineering Research Center for Biomanufacturing of Chiral Chemicals, College of Biotechnology and Bioengineering Zhejiang University of Technology Hangzhou China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Daifu","family":"Shi","sequence":"additional","affiliation":[{"name":"Key Laboratory of Bioorganic Synthesis of Zhejiang Province, The National and Local Joint Engineering Research Center for Biomanufacturing of Chiral Chemicals, College of Biotechnology and Bioengineering Zhejiang University of Technology Hangzhou China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Weibin","family":"Shu","sequence":"additional","affiliation":[{"name":"Key Laboratory of Bioorganic Synthesis of Zhejiang Province, The National and Local Joint Engineering Research Center for Biomanufacturing of Chiral Chemicals, College of Biotechnology and Bioengineering Zhejiang University of Technology Hangzhou China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Shuping","family":"Zou","sequence":"additional","affiliation":[{"name":"Key Laboratory of Bioorganic Synthesis of Zhejiang Province, The National and Local Joint Engineering Research Center for Biomanufacturing of Chiral Chemicals, College of Biotechnology and Bioengineering Zhejiang University of Technology Hangzhou China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0370-2502","authenticated-orcid":false,"given":"Yaping","family":"Xue","sequence":"additional","affiliation":[{"name":"Key Laboratory of Bioorganic Synthesis of Zhejiang Province, The National and Local Joint Engineering Research Center for Biomanufacturing of Chiral Chemicals, College of Biotechnology and Bioengineering Zhejiang University of Technology Hangzhou China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yuguo","family":"Zheng","sequence":"additional","affiliation":[{"name":"Key Laboratory of Bioorganic Synthesis of Zhejiang Province, The National and Local Joint Engineering Research Center for Biomanufacturing of Chiral Chemicals, College of Biotechnology and Bioengineering Zhejiang University of Technology Hangzhou China"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"311","published-online":{"date-parts":[[2025,11,4]]},"reference":[{"key":"e_1_2_10_2_1","doi-asserted-by":"publisher","DOI":"10.1002\/prot.22922"},{"key":"e_1_2_10_3_1","doi-asserted-by":"publisher","DOI":"10.1110\/ps.034199.107"},{"key":"e_1_2_10_4_1","doi-asserted-by":"publisher","DOI":"10.1021\/acs.jafc.3c09217"},{"key":"e_1_2_10_5_1","doi-asserted-by":"publisher","DOI":"10.1021\/acssuschemeng.3c02833"},{"key":"e_1_2_10_6_1","doi-asserted-by":"publisher","DOI":"10.1089\/rej.2018.2077"},{"key":"e_1_2_10_7_1","doi-asserted-by":"publisher","DOI":"10.1038\/s41598-019-46120-z"},{"key":"e_1_2_10_8_1","doi-asserted-by":"publisher","DOI":"10.1038\/s41580-020-00313-x"},{"key":"e_1_2_10_9_1","doi-asserted-by":"publisher","DOI":"10.2174\/0929867311320100006"},{"key":"e_1_2_10_10_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.neuron.2021.02.009"},{"key":"e_1_2_10_11_1","doi-asserted-by":"publisher","DOI":"10.1021\/ja300897h"},{"key":"e_1_2_10_12_1","doi-asserted-by":"publisher","DOI":"10.1128\/jb.187.8.2774-2782.2005"},{"key":"e_1_2_10_13_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.foodchem.2022.134824"},{"key":"e_1_2_10_14_1","doi-asserted-by":"crossref","unstructured":"Jaemoon Lee H. 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